The invention pertains to the detection and isolation of BRCA1. More particularly, the invention pertains to the detection and isolation of BRCA1 found in milk fat globule membranes.
It has been recently demonstrated that two genes, namely, BRCA1 encoding a 220 kDa protein, and BRCA2 encoding an approximate 420 kDa protein, are involved in hereditary breast and ovarian cancers. The BRCA1 protein contains an N-terminus a zinc finger domain, i.e. the region which can bind to DNA. The BRCA1 protein may represent one of the transcription factors, playing an important role in the differentiation of mammary gland cells. The C-terminal end of the BRCA1 is essential to normal BRCA1 function in breast epithelial cells, because patients inheriting 1853Stop develop very early onset breast cancer. The development of hereditary breast cancers can be seen as the result of mutations or deletions of the BRCA1 gene leading to the production of altered forms (truncated) of BRCA1 protein which cannot function as suppressors of cell growth (tumor suppressors).
It has been shown that the BRCA1 protein also can be involved in sporadic breast cancers. In this case, the transport of BRCA1 protein into the nucleus of cell is believed to be altered. Therefore, an accumulation of BRCA1 protein occurs in the cytoplasm of mammary gland secretory cells. Although there is some controversy regarding this hypothesis, the result cannot be ignored. It has been reported that the expression of BRCA1 in sporadic cancers is diminished. Quite recently, it was reported that BRCA1 is a secreted protein. Clustered BRCA1 proteins were detected by immunogold electron microscopy in small membrane bound vesicles in the apical cytoplasm of mammary epithelial cells. It is suggested that BRCA1 can manifest its function through the secretion and subsequent binding to the putative receptor of the same cell.
Though there is no consensus on the mechanism of action of BRCA1 we have to accept that BRCA1 is an important protein for development and differentiation of mammary gland secretory epithelial cells. Mutations or microdeletions of the BRCA1 gene or altered expression of BRCA1 mRNA and BRCA1 protein can lead to dedifferentiation with possible formation of cancer cells.
Recently, the complete sequence of the BRCA2 gene was reported. This gene encodes the protein of 3418 amino acids i.e. this protein would be about 420 kdas. Biochemical function of BRCA2 is not yet clear though the presence of regulatory signals are indicated. A mutational profile of BRCA2 differs from BRCA1 and is characterized by microdeletions rather than point mutations. The microdeletions in BRCA2 gene would explain the truncated forms of the BRCA2 protein. The 15 mutations observed so far by the Myriad group are quite distinct. This situation can complicate the development of the genetic test for the determination of predisposition to breast cancer. BRCA2 as BRCA1 has a sequence (xe2x80x9cgranin consensusxe2x80x9d) which is typical for a number of secretory proteins. The secretion of BRCA2 protein by mammary epithelial cells still has to be determined.
The protein BRCA1 is produced in mammary cells, but isolating BRCA1 from mammary cells is expensive and time consuming, such that it is entirely impractical to isolate commercial quantities of BRCA1. Therefore, it will be extremely important to develop an inexpensive method of isolating BRCA1 if it is to be used as a treatment.
One of the remarkable features of milk is its content of lipid droplets coated with proteo-lipid material. These droplets are milk fat globule membranes (MFGM). The MFGM, is composed of four layers: the thin membrane possibly derived from intracellular lipovesicles; the protein coat; the lipid bilayer, primarily derived from the apical plasma membrane and possibly secretory vesicle membranes; and the glycocalyx. Electron microscopy of the MFGM revealed that a major component of it represents membranous sheets with associated coat material; however, some MFGM also appeared as vesicles with little or no coat material. It is very likely that the synthesis of milk proteins during lactation is simultaneously accomplished by intensive synthesis of the above mentioned membrane components needed to replenish their loss by their extrusion from mammary gland secretory cells. In this sense, milk is a unique depo of the biological membranes synthesized inside the mammary secretory epithelial cells.
The methods of the present invention arose from the discovery that the protein encoded by the BRCA1 gene is found in the milk fat globule membranes from humans and cows. Therefore, BRCA1 protein can be isolated from milk produced by lactating animals.
The level of expression of BRCA1 can be determined by sampling the levels of BRCA1 protein found in the MFGM of lactating animals. The present invention also includes a method of determining the likelihood that a woman will develop breast cancer by measuring the amount of BRCA1 expression during lactation. The detection of expression of BRCA1 can be accomplished with an antibody raised specifically against BRCA1 protein, by isolating BRCA1 from the milk fat globule membranes or by detecting activity of BRCA 1. The level of BRCA1 is compared to a reference scale of propensity for breast cancer development correlated to normally active BRCA1 levels.
The present invention also includes a method of providing BRCA 1, BRCA2 and fatty acid binding proteins for oral consumption. The MFGM can be isolated from milk and provided in a form suitable for oral consumption, i.e. a tablet or capsule of separated milk fat globule membranes or a food additive.